The molecular structures of 3 flavocytochromes will be studied and compared, utilizing the technique of x-ray crystallography. Functional derivatives, site-directed mutants and other closely-related proteins will be studied. Two aspects of the systems under study are of particular interest, the mechanisms of flavin-linked substrate oxidation and the subsequent electron transfer steps between flavin and heme cofactors. Flavocytochrome b2 is a tetramer of Mr 230,000 located in yeast mitochondria. Each subunit consists of a cytochrome domain and a flavin- binding domain containing non-covalently bound heme and FMN, respectively. Its structure is known at 2.4 A resolution and several mutant forms affecting its catalytic and electron transfer properties will be studied, along with isolated flavin domain and closely-related flavoprotein oxidase. p-Cresol methylhydroxylase is an alpha2Beta2 dimer of Mr 115,000 found in Pseudomonas putida. One type of subunit contains FAD covalently bound to a tyrosine side chain, while the other type is a c-type cytochrome. Its structure has recently been refined at 2.5 A resolution and indicates several key residues at the active site and the flavin-cytochrome interface. Studies of functional derivatives and mutant forms of the enzyme are planned. Flavocytochrome c552 is an alphabeta dimer of Mr. 67,000 found in the phototrophic bacterium Chromatium vinosum. One subunit contains FAD covalently bound to a cystein side chain while the other is a diheme cytochrome. A chain tracing has recently been obtained from a 3.0 A resolution electron density map. The flavoprotein subunit closely resembles glutathione reductase. One of the hemes of the cytochrome is about 10 A from the flavin ring. Studies of bound inhibitors and substrates and eventually site-directed mutants are planned.